High mobility group (HMG) proteins HMGI, HMGY, HMGI-C, and Chironomus HMGI are DNA-binding proteins thought to modulate the assembly and the function of transcriptional complexes. Each of these proteins contains three DNA-binding domains (DBD), properties of which appear to be regulated by phosphorylation. High levels of these proteins are characteristic for rapidly dividing cells in embryonic tissues and tumors. On the basis of their occurrence, specific functions for each of these proteins have been postulated. In this study we demonstrate differences in the nature of contacts of these proteins with promoter region of the interferon-␤ gene. We show that HMGI and HMGY interact with this DNA via three DBDs, whereas HMGI-C and Chironomus HMGI bind to this DNA using only two domains. Phos- Despite continuously increasing interest in the function(s) of this group of proteins, the nature of the interaction of these proteins with DNA is still not sufficiently understood. The proteins of the HMGI/Y family are 10 -11 kDa in size, are highly charged, are rich both in acidic and basic residues, are proline-rich, and contain only few residues with bulky hydrophobic side chains. This unusual amino acid composition inhibits folding of the polypeptide backbone of these proteins into any defined secondary structure. Common for HMGI/Y proteins is the presence of three putative DNA-binding domains (DBD), so called . NMR analysis of a complex of a peptide derived from HMGI(Y) bound to a short DNA fragment revealed that the centrally located RGR residues are essentially for binding and responsible for contacts of the protein with the bases and phosphate-sugar backbone (27). Alternative approaches, which used deletion and point-mutated proteins, revealed that two or three DBDs of the protein bind to DNA in a cooperative way (28 -30). Application of the protein-footprinting method for mapping of protein regions interacting with DNA (31) allowed more detailed characterization of the binding of HMGI(Y) proteins to DNA (32). A combination of this method

The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dAdT)⅐poly(dA-dT) and a fragment of the interferon-␤ promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin.

A splicing factor SF2/ASF is a natural substrate for the kinase activity of human topoisomerase I. This study demonstrates that SF2/ASF inhibits DNA cleavage by human topoisomerase I induced by the anti-cancer agent camptothecin. The inhibition is independent of the phosphorylation status of SF2/ASF. We show that the inhibition did not result from binding of SF2/ASF to DNA that would hinder interactions between topoisomerase I and DNA. Neither it was a consequence of a loss of sensitivity of the enzyme to camptothecin. We provide evidence pointing to reduced formation of the cleavable complex in the presence of SF2/ ASF as a primary reason for the inhibition. This effect of SF2/ASF is reflected by inhibition of DNA relaxation catalysed by topoisomerase I.Keywords: topoisomerase I; SF2/ASF; camptothecin.Eukaryotic topoisomerase I (topo I) catalyses DNA relaxation and plays a key role in DNA replication, transcription and recombination in the cell [1]. Topo I is also a target for several anti-cancer drugs derived from the cytotoxic plant alkaloid camptothecin [2]. A transient intermediate of the enzyme's catalytic cycle is the cleavable complex, consisting of the enzyme linked covalently to one strand of DNA. This complex is stabilized by camptothecin and can be detected as topo I-associated DNA single-strand breaks [3]. These breaks are usually called camptothecin-induced DNA cleavage. Camptothecin-induced DNA cleavage is essentially reduced by binding of SV40 T antigen [4] or ATP [5] to topo I. The complex is not detected for several mutated forms of the enzyme that are resistant to camptothecin (reviewed in [6]). Sensitivity to camptothecin is also diminished by dephosphorylation of topo I [7,8].Human topoisomerase I (htopo I) possesses a protein kinase activity which is specific towards serine residues of splicing factors containing a serine-arginine (SR) motif [9]. Phosphorylation of SR proteins is instrumental for the recruitment of these proteins to active sites of transcription in vivo [10] and for their activity as splicing factors [11]. SF2/ ASF is the main splicing factor containing an SR motif [12] phosphorylated by htopo I/kinase [9]. The binding site for SF2/ASF in htopo I is located between residues 135 and 175 [13]. This region is included in the N-terminal domain of the enzyme that in general is dispensable for both relaxation and binding of camptothecin [14,15]. However, detailed studies have recently revealed that the N-terminal domain influences the rate of relaxation and sensitivity of the enzyme to camptothecin [16].In the present work we report that the natural substrate for the kinase activity of htopo I, SF2/ASF protein, inhibits camptothecin-induced DNA cleavage by the enzyme. This effect results from reduced amount of the cleavable complex formed by htopo I in the presence of SF2/ASF. M A T E R I A L S A N D M E T H O D S Plasmids and strainsThe pRS426-based plasmid, containing complete human topo I cDNA under the control of the GAL1-10, was a gift from B. R. Knudsen (University of Aarhu...

High-mobility group I/Y (HMGI/Y) proteins are chromosomal proteins involved in gene and chromatin regulation. Elevated levels of HMGI/Y proteins were reported in diverse malignant tumors, and rearrangements of their genes are casually involved in the development of benign tumors. In humans, the chromosomal locus Xp22 has been often found to be affected in diverse benign mesenchymal tumors. Recent studies revealed that this region contains a retropseudogene HMGIYL1 which potentially can be activated in a way of "exonization" upon aberrations involving this region. The coding sequence of the HMGIY-L1 is highly homologous to the HMGI(Y) gene. On the protein level, both HMGIYL1 and HMGI differ at few amino acid residues, including their putative DNA-binding domains (DBDs). Here we have approached the question of whether the HMGIYL1 product would be able to adopt a role of HMGI in the context of binding to gene promoters and chromatin. Comparative binding studies, employing protein footprinting technique, revealed that HMGIYL1 has lost the ability to bind to the promoter of the interferon beta gene, but retained its high affinity for the four-way junction DNA. Our results stress the importance of particular residues within the DBDs for DNA binding and demonstrate that tight binding of HMGI/Y proteins to the four-way junction DNA can be achieved in alternative ways. The binding of HMGIYL1 to four-way junction DNA suggests that activation of the HMGIYL1 gene would yield a protein sharing some binding properties with HMG1-box proteins and histone H1. Thus, the HMGIYL1 could interplay together with these components in chromatin regulation.

Phage display procedure was applied to the N-terminal domain of human topoisomerase I. The consensus sequence identified for clones binding to the N-terminal domain was found in 35 human proteins that are either permanently or temporarily located in the nucleus. They are in majority involved in the DNA repair, transcription, RNA metabolism or cell cycle control. Four of identified proteins: Bub3 protein, Cockayne syndrome protein A, damaged DNA binding protein 2 and GRWD protein belong to WD-repeat proteins and their sequences recognized by the N-terminal domain are identically localized.